The long-term goals of this project are to define the sensitivity of vagal afferent neurons to stimuli that are important to the control of energy homeostasis and to determine the cellular mechanisms by which these stimuli activate this afferent signaling pathway. The investigations in this proposal will specifically focus on the mechanisms of activation and degree of interaction of cholecystokinin (CCK), leptin, and free fatty acids (FFA) on vagal afferent neurons. These substances were chosen because there is abundant evidence that CCK, leptin, and FFA each participates in the control of body energy balance, and evidence exists that there are systemic interaction between these substances. Published reports, as well as preliminary data presented in the proposal, indicate that subpopulations of vagal afferents are sensitive to CCK, leptin, and FFA. Preliminary results further indicate that there are important interactions between these substances in the activation of individual vagal afferent neurons. In this project experiments will concentrate primarily on vagal afferent neurons isolated from adult rat nodose ganglia. The neurons in this preparation retain most, if not all, of the properties ascribed to intact vagal afferent fibers in vivo. The use of this preparation enables the design of much more refined and tightly controlled electrical, chemical, and pharmacological investigations of neuronal responses than are possible in vivo. This preparation will be used to address three specific aims: 1) A combination of single cell Ca2+ imaging, retrograde labeling, and immunohistochemistry, will be utilized to establish the innervation targets of discrete vagal afferent populations sensitive to CCK, leptin, and FFA. 2) Pharmacological tools combined with patch clamp electrophysiology and Ca2+ imaging will be utilized to determine the cellular mechanisms by which CCK, leptin, and FFA activate vagal afferent neurons, and to determine the cellular mechanisms by which these individual stimuli interact at the level of the vagal afferent neuron. 3) The hypothesis that in addition to acute activation and interactive effects, leptin, CCK, and FFA also have chronic and enduring effects on the responsiveness of these neurons will be tested. The degree to which individual vagal afferent neurons respond to and integrate signals from these disparate processes largely remains unappreciated. The insights developed in this series of investigations will enable a better reconstruct of the systemic actions and interactions of these important regulatory signals. A better appreciation of this important signaling pathway will contribute to the understanding of energy homeostasis and GI function, and aid in the design of therapeutic interventions for better health.